A novel microchip is customized to answer specific questions and has oligonucleotides positioned on the microchip so that multiple bits of information are evidenced to a simpler pattern A new method of hybridization to a microchip is also presented.
Differences in nucleotide and amino acid sequences may be exploited to analyze environmental, food or biological samples. Detection and identification of microorganisms is important for clinical purposes and for determination of contaminated food, air, water or soil. Studies in environmental microbiology are often limited by the inability to unambiguously identify and directly quantify the enormous diversity of natural populations. This problem is now changing with increasing use of molecular techniques to directly measure different genetic features. (Mobarry et al., 1996; Stahl, 1995; Wagner et al., 1995) For example, DNA probes are now commonly used to detect by hybridization, genes encoding proteins involved in specific catabolic functions, and to resolve different genetic populations in the environment. In particular, the use of group-specific DNA probes complementary to the small subunit (SSU) 16S rRNA has provided a comprehensive framework for studies of microbial population structure in complex systems. Sequencing of this subunit revolutionized microbial classification and led to the discovery of archebacteria. (Woese, 1987) A large number of the sequences for different organisms has been collected. (Maidak et al., 1996) Every microorganism species is characterized by a specific DNA sequence within a variable region of its ribosomal RNA gene or other genes. A highly efficient procedure for microorganism classification and for construction of their evolutionary trees is based on these observations. Identification of specific sequences in ribosomal DNA is a reliable microbial analysis that can be carried out by direct DNA sequencing. However DNA sequencing is a rather complicated, expensive and time consuming procedure to use for serial microbial analysis on a commercial scale for environmental or medical applications. Consequently, new methods are needed to make sequence matching commercially feasible.
Also, methods are needed that are transportable to the field. A nucleic acid hybridization is a highly specific and sensitive procedure that allows a specific sequence to be detected and identified among other millions of sequences in a genome of higher organisms, or among a mixture of different organisms. The principle of hybridization is that sequences hybridize as a function of the similarity of their linear nucleotide sequence. The hybridization of DNA or RNA extracted from even a very complicated mixture to a specific oligonucleotide probe has resulted in unambiguous identification of specific microorganisms in an environmental sample, for example. In the course of such an analysis, RNA or DNA is extracted from a sample of microorganisms isolated from water solutions, air or soil, immobilized on a filter and then hybridized successively with several oligonucleotide probes for different microorganisms. However, for this purpose, the sample needs to be checked for the presence of hundreds or thousands of different oligonucleotides corresponding to various microorganisms which is prohibitively laborious and expensive using present methods and yields results that must be interpreted by a computer in order to decipher the identification. What is needed is a simplified pattern to provide rapid answers to specific questions, e.g. are any known pathogens in a water sample?
The scope of applications of nucleotide hybridization is often limited by the nature of the assays, generally involving the independent hybridization and interpretation of multiple environmental samples to multiple DNA probes. In addition, some detection assays require amplification of the target nucleic acid, for example, via PCR. This may contribute to quantitative biases. Thus, there is need for assays that provide for greater sample through-put capacity and greater sensitivity, rapid read-out of results.
Another area in which specific DNA or RNA sequences are of interest is mutation and polymorphism analyses. The number of base changes discovered (mutations) in different genes is growing rapidly. These changes are associated with genetic diseases, with disease predispositions and cancers, with development of drug resistance in microorganisms, and with genetic polymorphisms. Polymorphisms are useful for determining the source of a sample, e.g. in forensic analyses. Polymorphisms such as in the HLA system are essential to predict success of tissue transplants. The ability to simultaneously analyze many mutations in a gene in a simple, fast, and inexpensive way is essential in clinical medicine and this need has stimulated the development of different methods for screening mutations, but all have serious limitations. What is needed are kits that are transportable and interpretable, e.g. for use in clinics without high technology microscopes.
Hybridization of filter-immobilized DNA with allele-specific oligonucleotides was suggested as a way to screen for mutations. (Conner et al., 1983) However, the number of alleles that can be assayed at one time is limited, the filters are usable only for a few times, and there is little opportunity for complex analysis or easy interpretation of results.
A possible solution to large scale hybridization is to use microchips for DNA sequence hybridizations (SHOM, sequencing by hybridization with oligonucleotides in a microchip) (e.g. Khrapko, 1996; Yershov, 1996). The development of an array of hundreds or thousands of immobilized oligonucleotides, the so-called xe2x80x9coligonucleotide chipsxe2x80x9d, permits simultaneous analysis of many mutations (for a review, see Mirzabekov, 1994). Such arrays can be manufactured by a parallel synthesis of oligonucleotides (Southern et al., 1992; Fodor et al., 1991; Pease et al., 1994; Matson et al., 1995) or by chemical immobilization of presynthesized oligonucleotides (Khrapko et al., 1991; Lamture et al., 1994; Ghu et al., 1994). Glass surfaces (Southern et al., 1992; Fodor et al., 1991; Ghu et al., 1994), glass pores (Beattie et al., 1995), polypropylene sheets (Matson et al., 1995), and gel pads (Khrapko et al., 1991; Yershov et al., 1996) have been used as solid supports for oligonucleotide immobilization. However xe2x80x9cOligonucleotide array technology has not yet lived up to its promise.xe2x80x9d Southern, 1996 p. 115.
Some of the deficiencies in the art are unpredictability of the results, lack of knowledge of optimum conditions, and failure to demonstrate accuracy and commercial feasibility. Moreover, analysis of the results of hybridization requires computer programs capable of assimilating and interpreting multiples bits of information, and high technology microscopes. The microchips are neither portable, reusable, nor easily interpreted.
This invention embodies applications of oligonucleotide microchip technology wherein the microchip is a biosensor and customized oligonucleotide microchips are designed for specific applications of nucleic acid hybridization.
Hybridization is a process by which, under defined reaction conditions, partially or completely complementary nucleic acids are allowed to join in an antiparallel fashion to form specific and stable hydrogen bonds.
Aspects of the invention include:
1. microchips designed so that multiple bits of genetic information are converted to a pattern, which is interpreted as a unit, wherein the appearance of the pattern provides answers to specific questions; this construction facilitates providing easily interpretable answers provided by hybridization patterns and removes some need for high technology instruments to interpret the results of hybridization; and
2. improved methods of hybridizing oligonucleotides in a sample to oligonucleotides on a customized microchip do not require a washing step but rather measure non-equilibrium melting curves (temperature curves) that do not require washing with a solution that removes immobilized oligonucleotides from microchips; this means that microchips are reusable because the oligonucleotides anchored within the gel elements, do not wash away, and are available for reuse. (Microchips with samples are generally kept in solution, however, microchips can be dried and stored for many months before being reused.)
The patterns exhibited after hybridization to a microchip generally are not directly related to the nature of the hybridizations and are not simply converting a xe2x80x9cyesxe2x80x9d or a xe2x80x9cnoxe2x80x9d signal, or a xe2x80x9cpositivexe2x80x9d or xe2x80x9cnegativexe2x80x9d signal to a binary outcome, nor are the patterns of the present invention converting a gradation of quantities to another form of gradation, e.g. colorimetric gradations. The deliberate organization of the oligonucleotides on the microchips themselves does not transmit information; only after hybridization with a test nucleic acid will the hybridization signal itself form the pattern. The pattern is then detected by a detection means which can include visual interpretation without the aid of additional detection instrumentation.
By choosing ordered schemes of oligonucleotide positioning on the microchips, visual signals are simplified and enhanced, e.g. the letter xe2x80x9cPxe2x80x9d is observed if certain pathogenic groups are present; columns of gel elements on the chip that include the same oligonucleotide probes, will be readily detectable as a positive linear column, if the matching oligonucleotides are in the test sample. The visual appearance may be strong enough to see with the naked eye, may be determined with a UPC (Universal Product Code or xe2x80x9cbar codexe2x80x9d) laser scanner, or with a laser gun. The wavelength of the scanner and the sensor that accepts the signal for a bar code must be concordant with the dye or label used to hybridize the DNA.
Of course, aspects one and two do not have to be used together. Designs that result from converting multiple amounts of genetic information obtained by large numbers of hybridizations of oligonucleotides to simpler, readily interpretable patterns, could be done on microchips constructed and analyzed by the methods used prior to the present invention.
Similarly, the improved methods of providing hybridization results on microchips could be used on microchips that are not designed to convert multiple pieces of genetic information into a simpler pattern.
Other aspects of the invention include improved predictability, increased accuracy, and standardized factors for detection and identification of nucleotide sequences. The improvements result from optimizing conditions, methods and compositions for microchip hybridization. Deliberate ordered schemes that are designed to answer specific questions and that convert complex data to simpler patterns, are followed so that much hybridization information can be readily obtained from a single scan of a microchip to detect hybridization of immobilized oligonucleotides by nucleic acids in a sample to be investigated. Samples include air, water, soil, blood, cells, tissue, tissue culture and a food. An aspect of the invention is that the same microchip can be used for hybridization for more than 20-30 times, without any noticeable deterioration of the hybridization signal because immobilized oligonucleotides are not washed out or stripped. Customized sets of microchips are obtained for specific applications. Also, parallel hybridization of nucleic acids in a sample to many oligonucleotides on a microchip is possible, allowing replication and standardization. For example, the sequence diversity of SSU rRNAs recovered from different microbial populations of varying abundances is analyzed by a single hybridization to a microchip. A large number of HLA alleles, are assayed by a single hybridization to a microchip.
The invention relates a method for identifying a nucleotide sequence in a sample using a microchip, said method comprising:
a) providing a customized matrix of oligonucleotides on the microchip designed to identify genetic sequences in the sample, wherein an ordered scheme positions oligonucleotides to provide a pattern to answer specific questions after hybridization;
b) hybridizing nucleic acids extracted from the sample as such or after amplification on said microchip; and
c) identifying the nucleotide sequences represented in said sample by analyzing the pattern of the oligonucleotides which hybridized to the sequences, said pattern provided by signals.
The nucleic acids suitable for the practice of the invention include DNA, mRNA, 16S rRNA sequences and other RNA species.
Customized oligonucleotide microchips are aspects of the invention. The microchip includes a gel-matrix affixed to a support, said matrix is formed by a plurality of gel pad element sites. The number of sites is determined by the number of oligonucleotides in the array. Each gel element contains one chemically immobilized oligonucleotide of a desired sequence, length and concentration; the gel elements being separated from one another by hydrophobic glass spaces and the gel portions having a vertical height above the plane of the interstitial spaces of generally not more than 30 xcexcm. In some applications, the same type of oligonucleotides may be immobilized to different gel pads to form a pattern.
The invention relates screening nucleic acid preparations for genes, RNA transcripts or any other unique nucleotide sequences, for example those that encode microbial 16S ribosomal RNAs. Ratios of DNA/RNA or any other unique nucleotide sequences specific for certain types of organisms are suitable. Multiple labeling allows simultaneous detection and quantitative comparison of different nucleic acid sequences that are hybridized to a microchip.
The methods of the present invention include labeling the oligonucleotide sequence in said sample before bringing it in contact with the array. A suitable label is a fluorescent dye. A plurality of different dyes may be used concurrently. Oligonucleotides immobilized on a customized microchip include those complementary to the beta globin gene, sequences specific for Salmonella, or polymorphic HLA allele sequences.
An oligonucleotide microchip for the detection and classification of nitrifying bacteria has a customized design wherein identifying labels in the cells of the microchip refer to oligonucleotides selected from a class of bacteria, and the selection is designed to answer specific questions regarding classification.
An embodiment of an application of the present invention is detecting and identifying microorganisms in samples obtained from the environment, e.g. water, air or soil samples to check for pollutants; biological samples obtained for medical diagnosis; or food samples to check for contamination. Other applications include forensic testing to identify DNA in samples obtained for criminal investigations, and detection of chromosomal fragments, or single gene mutations e.g. for diagnosing genetic diseases such as xcex2-thalassemia or types of cancers. Tissue typing for polymorphic HLA alleles for transplantation or studying human diversity is facilitated.
The nucleic acid preparations are made from samples collected in any type of environment, where detection and identification of the microorganisms in that environment is of interest, or where it is likely that new (previously unidentified) organisms may be discovered.
DNA and RNA molecules in a sample can be separated from each other during their isolation and labeled with different fluorescent dyes. These RNA and DNA molecules are simultaneously hybridized with oligonucleotides on a microchip that is specific to the sample to be tested. The quantitative monitoring of the simultaneous hybridization of differently labeled DNA and RNA with a microscope that can discriminate multicolors at several wave lengths allows the calculation of DNA/RNA ratios in the sample. For bacterial samples, this ratio determines the state of vitality and physiological activity of the bacterium. In an embodiment, the ratio of RNA/DNA is used to discriminate the dead bacterium cells and spores from the active state of microbial growth. In the same way, a DNA or RNA molecule of a bacterial strain stained with one dye can be added in a calculated amount as an internal standard to a sequence or sequences under investigation in which the sequences being investigated stained with a different (second) dye. The fluorescence measurements of hybridization intensities at different wave lengths for the standard and investigated sequences (probes) allow relative quantitative ratios to be determined.
Hybridization on microchips allows unambiguous typing of different groups of chosen bacteria in a sample. Microchip hybridization is a simple, fast, inexpensive and reliable method for bacterial typing.
An aspect of the invention is that there is no limitation on the number of sequences that can be checked or the number of types of microorganisms that can be detected. Instead of multiple sequential hybridizations with different probes of, e.g. a 16S rRNA preparation, only one round of hybridization is required to find out what different sequences are in a sample. The volume of hybridizations is dramatically reduced and the assay requires much less RNA or DNA compared with standard techniques. An advantage is that culturing of bacteria and gene amplification can be avoided.
Methods of the invention significantly reduce sample preparation time, avoid the culturing of organisms collected from field situations, and allow the identification of all species of microorganisms contained in a particular sample. Portable microchips are available for field work.
For example, oligonucleotides complementary to small subunit rRNA sequences of selected microbial groups, encompassing key genera of nitrifying bacteria, were shown to selectively retain or hybridize with labeled target nucleic acid derived from either DNA or RNA forms of the target sequences. Methods and compositions of the present invention discriminate among the Genera, Nitrosomonas, Nitrobacter and Nitrosovibrio sp. using fluorescently labeled nucleic acid probes that hybridize to 16S rRNA sequences. Each species has specific DNA sequences within the variable region of its rRNA genes. Since the rRNAs are naturally amplified, often present in thousand of copies per cell, they provide greater sensitivity, eliminating the need for amplification in many applications.
The invention facilitates identification of organisms from environmental samples in a faster, and more economical approach than presently available. In addition, new species may be discovered that would be highly informative regarding taxonomic status of known as well as newly discovered organisms.
A diagnostic assay of the present invention for a mutation in a gene, includes the following steps:
a. designing a customized oligonucleotide microchip biosensor comprising oligonucleotides that hybridize to a gene having the mutation, wherein the oligonucleotides are positioned on the microchips so that patterns result depending on what oligonucleotides are in the sample to answer a specific question(s);
b. contacting a nucleic acid sample to the customized oligonucleotide microchip biosensor under conditions that allow hybridization of the nucleic acid to the microchip; and
c. determining the pattern of hybridization from which observation the presence of specific nucleic acid sequences is inferred and the specific question is answered.
For diagnostic assays for genetic diseases, sequence analysis of DNA is carried out by hybridization of PCR amplified DNA or its RNA transcripts with oligonucleotide array microchips. Polyacrylamide gel pads containing allele-specific immobilized oligonucleotides are fixed on a glass slide of the microchip. The RNA transcripts of PCR-amplified genomic DNA are optionally fluorescently labeled by enzymatic or chemical methods and hybridized with the microchip. In the field, the chemical methods are preferred because results are obtained faster, and some chemicals will fragment DNA at the same time which is needed for the sample.
When melting curve experiments are performed, both matching and mismatching oligos can be immobilized in the gel pads, and both matching and mismatching nucleic acids can be in the sample. The biochips are reusable in two types of embodiments: 1) the sample or test nucleic acids can be removed or stripped off the chip and a different test sample can be introduced and 2) the same melting point curve experiments can be run and re-run without any washing.
When experiments are performed with a different test sample, the original sample is removed from the chip by a washing or stripping procedure using distilled water at 60xc2x0 C. with an hour (or up to overnight) incubation. If the melting curve experiments are repeated (or reused) then the same sample is left in contact with the chip and appearance and disappearance of hybridization signal is observed over a variety of temperatures, usually ranging from 0xc2x0-50xc2x0 C.
When the chips are incubated, in order to remove the sample nucleotides, virtually none of the immobilized oligos are removed in the process. This is because the oligos are covalently linked to the gel matrix of the gel pads that form the microchip.
Repeated reuse of the chips in which different samples are applied after sequential removal is usually limited to about 50 uses, because eventually the amount of non-specific or background hybridization signal is greater than one-tenth of a mismatch hybridization signal. The conditions under which a chip would not be reusable (up to 50 times) are very few. Such conditions include allowing the chips to be cooled to xe2x88x9220xc2x0 C. or performing experiments where the chips are heated to above 70xc2x0 C., conditions that have been shown to cause degradation of the chips, thus rendering them unstable.
The simultaneous measurement in real time of the hybridization and melting curves on the entire oligonucleotide array is carried out with a fluorescence microscope with a laser light source equipped with CCD camera or a special laser scanner. Some work only with dried microchips. The monitoring of the hybridization specificity for duplexes with different stabilities and AT content is enhanced by its measurement at optimal discrimination temperatures on melting curves. Microchip diagnostics are optimized by choosing the proper allele-specific oligonucleotides from among the set of overlapping oligomers. The accuracy of mutation detection can be increased by simultaneous hybridization of the microchip with at least two differently labeled samples of normal and mutated alleles, and by parallel monitoring their hybridization with a multi-wavelength fluorescence microscope. The efficiency and reliability of the sequence analysis was demonstrated by diagnosing xcex2-thalassemia mutations and HLA polymorphisms. Determining levels of gene expression is an aspect of the invention.
Because the methods of the present invention require only a simple procedure of hybridization and because only one round of hybridization is necessary, it is fast and inexpensive. Because the invention allows a lot of information to be obtained from one experiment, in a simple pattern as compared to the analysis of hundreds of data points, it has increased efficiency. The invention is reliable because the microchips are reusable. Immobilized oligonucleotides are not washed out. There is no waste of hybridization probes, therefore the microchip hybridization is inexpensive and non-isotopic detection simplifies all procedures.
Effective and precise sequence analysis by the hybridization of a probe with rather short microchip-immobilized oligonucleotides depends on many factors. Major factors are the reliability of the discrimination of perfect duplexes from duplexes containing mismatches, differences in stability of AT- and GC-rich duplexes, the efficiency of the hybridization, and simplicity in the preparation of the labeled samples for hybridization.
Identification of base variations is significantly improved by parallel measuring of the melting curves of the duplexes formed on the entire oligonucleotide array, as well as by monitoring the simultaneous hybridization of two differently labeled samples at two wavelengths and by choosing proper allele-specific oligonucleotides.
Other factors to be considered for operation of the invention include (1) regulating the flow of the fluid containing a sample to be tested over the microchip during the hybridization; and (2) control of the temperature of the microchip gel layer and the fluid layer, in a differential manner, by placing a cooling and heating apparatus adjacent to the gel layer and the top fluid layer. The gel layer temperature is controlled in a uniform or gradient manner by a heating/cooling device attached to the glass plate substrate of the gels. For field work, the optimum temperature for a particular question is determined previously in a laboratory.
A definition of xe2x80x9ccustomized microchipxe2x80x9d is a microchip of gel elements on a support, wherein the oligonucleotides are immobilized in gel elements according to an ordered scheme such that multiple bits of information are ordered to a simpler pattern to answer a specific question.
Removal of test or sample nucleic acids from microchip is accomplished by an incubation step carried out using distilled water for at least one hour (up to overnight) at 60xc2x0 C. (This procedure is analogous to the step of xe2x80x9cstrippingxe2x80x9d a filter for re-use in the standard technique of probing a Southern blot.) The immobilized oligonucleotides in the gel matrix are not removed by this incubation as the oligonucleotides are covalently linked to the gel substrate.